Nucleic acid probes to mycobacterium tuberculosis

ABSTRACT

Hybridization assay probes specific for members of the Mycobacterium tuberculosis Complex and no other Mycobacterium species.

This application is a continuation of application Ser. No. 07/876,283now abadoned, filed Apr. 28, 1992.

FIELD OF THE INVENTION

The inventions described and claimed herein relate to the design andconstruction of nucleic acid probes for Mycobacterium tuberculosisComplex (TB Complex) which are capable of detecting the organisms intest samples for, e.g., sputum, urine, blood and tissue sections, food,soil and water.

BACKGROUND OF THE INVENTION

Two single strands of deoxyribo- ("DNA") or ribo-("RNA") nucleic acid,formed from nucleotides (including the bases adenine (A), cytosine (C),thymidine (T), guanine (G), uracil (U), or inosine (I)), may associate("hybridize") to form a double stranded structure in which the twostrands are held together by hydrogen ponds between pairs ofcomplementary bases. Generally, A is hydrogen bonded to T or U, while Gis hydrogen bonded to C. At any point along the chain, therefore, onemay find the classical base pairs AT or AU, TA or UA, GC, or CG. One mayalso find AG, GU and other "wobble" or mismatched base pairs.

When a first single strand of nucleic acid contains sufficientcontiguous complementary bases to a second, and those two strands arebrought together under conditions which will promote theirhybridization, double stranded nucleic acid will result. Underappropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids may beformed.

A probe is generally a single stranded nucleic acid sequence which iscomplementary to some degree to a nucleic acid sequence sought to bedetected ("target sequence"). It may be labelled with a detectablemoiety such as a radioisotope, antigen or chemiluminescent moiety. Abackground description of the use of nucleic acid hybridization as aprocedure for the detection of particular nucleic acid sequences isdescribed by Kohne, U.S. Pat. No. 4,851,330, and Hogan et al., EPOPatent Application No. PCT/US87/03009, entitled "Nucleic Acid Probes forDetection and/Or Quantitation of Non-Viral Organisms."

Hogan et al., supra, also describes methods for determining the presenceof RNA-containing organisms in a sample which might contain suchorganisms. These methods require probes sufficiently complementary tohybridize to the ribosomal RNA (rRNA) of one or more non-viral organismsor groups of non-viral organisms. The mixture is then incubated underspecified hybridization conditions, and assayed for hybridization of theprobe and any test sample rRNA.

Hogan et al. also describes probes which detect only specificallytargeted rRNA subunit subsequences in particular organisms or groups oforganisms in a sample, even in the presence of many non-relatedorganisms, or in the presence of the closest known phylogeneticneighbors. Specific examples of hybridization assay probes are providedfor Mycobacterium tuberculosis. Such probe sequences do not cross reactwith nucleic acids from other bacterial species or infectious agent,under appropriate hybridization stringency conditions.

SUMMARY OF THE INVENTION

This invention discloses and claims novel probes for the detection ofMycobacterium tuberculosis (TB) Complex. These probes are capable ofdistinguishing between the Mycobacterium tuberculosis Complex and itsknown closest phylogenetic neighbors. The Mycobacterium tuberculosisComplex consists of the following species: M. tuberculosis, M. bovis, M.bovis BCG, M. africanum, M. microti. These probes detect unique rRNA andgene sequences encoding rRNA, and may be used in an assay for thedetection and/or quantitation of Mycobacterium tuberculosis Complex.

Organisms of the TB Complex are responsible for significant morbidityand mortality in humans. M. tuberculosis is the most common TB Complexpathogen isolated from humans. M. bovis BCG may be transmitted frominfected animals to humans. M. africanum causes pulmonary tuberculosisin tropical Africa and M. microti primarily infects animals.

Tuberculosis is highly contagious, therefore rapid diagnosis of thedisease is important. For most clinical laboratories assignment of anisolate to the TB Complex is sufficient because the probability that anisolate is a species other than M. tuberculosis is extremely small. Anumber of biochemical tests are recommended to speciate members of theTB Complex if further differentiation is required.

Classical methods for identification of mycobacteria rely on stainingspecimens for acid fast bacilli followed by culture and biochemicaltesting. It could take as long as two months to speciate an isolateusing these standard methods. The use of DNA probes of this inventionidentifies TB Complex isolated from culture in less than an hour.

Thus, in a first aspect, the invention features a hybridization assayprobe able to distinguish Mycobacterium tuberculosis from otherMycobacterium species; specifically, the probe is an oligonucleotidewhich hybridizes to the rRNA of the species Mycobacterium tuberculosisat a location corresponding to 23 bases in the insert region beginningat the equivalent of base 270 of E. coli 23S rRNA, or to 21 bases in theinsert region beginning at the equivalent of base 1415 of E. coli 23SrRNA, or an oligonucleotide complementary thereto; that is, theoligonucleotide comprises, consists essentially of, or consists of thesequence (SEQ ID NO: 1) GGTAGCGCTGAGACATATCCTCC, or (SEQ ID NO: 2)CAGAACTCCACACCCCCGAAG, or oligonucleotides complementary thereto, withor without a helper probe, as described below.

By "consists essentially of" is meant that the probe is provided as apurified nucleic acid which hybridizes under stringent hybridizingconditions with the desired organism and not with other relatedorganisms. Such a probe may be linked to other nucleic acids which donot affect such hybridization. Generally, it is preferred that the probebe of between 15 and 100 (most preferably between 20 and 50) bases insize. It may, however, be provided in a vector.

In related aspects, the invention features a nucleotide polymer able tohybridize to the above oligonucleotides, a nucleic acid hybrid formedwith the above oligonucleotides, and a nucleic acid sequencesubstantially complementary thereto. Such hybrids are useful since theyallow specific detection of the TB complex organisms.

The probes of this invention offer a rapid, non-subjective method ofidentification and quantitation of a bacterial colony for the presenceof specific rRNA sequences unique to all species and strains ofMycobacterium tuberculosis Complex.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Probes

We have discovered DNA probes complementary to a particular rRNAsequence obtained from Mycobacterium tuberculosis. Furthermore, we havesuccessfully used those probes in a specific assay for the detection ofMycobacterium tuberculosis, distinguishing members of the M.tuberculosis complex from their known and presumably most closelyrelated taxonomic or phylogenetic neighbors.

We have identified suitable variable regions of the target nucleic acidby comparative analysis of rRNA sequences both published in theliterature and sequences which we have determined. Computers andcomputer programs which may be used or adapted for the purposes hereindisclosed are commercially available. Since the sequence evolution ateach of the variable regions (for example, spanning a minimum of 10nucleotides) is, for the most part, divergent, not convergent, we canconfidently design probes based on a few rRNA sequences which differbetween the target organism and its phylogenetically closest relatives.We have seen sufficient variation between the target organism and theclosest phylogenetic relative found in the same sample to design theprobe of interest.

We have identified the following useful guidelines for designing probeswith desired characteristics. Because the extent and specificity ofhybridization reactions such as those described herein are affected by anumber of factors, manipulation of one or more of those factors willdetermine the exact sensitivity and specificity of a particular probe,whether perfectly complementary to its target or not. The importance andeffect of various assay conditions, explained further herein, are knownto those skilled in the art.

First, the stability of the probe:target nucleic acid hybrid should bechosen to be compatible with the assay conditions. This may beaccomplished by avoiding long A and T rich sequences, by terminating thehybrids with G:C base pairs, and by designing the probe with anappropriate Tm. The beginning and end points of the probe should bechosen so that the length and %G and %C result in a Tm about 2-10° C.higher than the temperature at which the final assay will be performed.The base composition of the probe is significant because G-C base pairsexhibit greater thermal stability as compared to A-T base pairs due toadditional hydrogen bonding. Thus, hybridization involving complementarynucleic acids of higher G-C content will be stable at highertemperatures.

Conditions such as ionic strength and incubation temperature under whicha probe will be used should also be taken into account in constructing aprobe. It is known that hybridization will increase as the ionicstrength of the reaction mixture increases, and that the thermalstability of hybrids will increase with increasing ionic strength. Onthe other hand, chemical reagents, such as formamide, urea, DMSO andalcohols, which disrupt hydrogen bonds, will increase the stringency ofhybridization. Destabilization of the hydrogen bonds by such reagentscan greatly reduce the Tm. In general, optimal hybridization forsynthetic oligonucleotide probes of about 10-50 bases in length occursapproximately 5° C. below the melting temperature for a given duplex.Incubation at temperatures below the optimum may allow mismatched basesequences to hybridize and can therefore result in reduced specificity.

It is desirable to have probes which hybridize only under conditions ofhigh stringency. Under high stringency conditions only highlycomplementary nucleic acid hybrids will form (i.e., those having atleast about 14 out of 17 bases in a contiguous series of bases beingcomplementary); hybrids without a sufficient degree of complementaritywill not form. Accordingly, the stringency of the assay conditionsdetermines the amount of complementarity needed between two nucleic acidstrands forming a hybrid. Stringency is chosen to maximize thedifference in stability between the hybrid formed with the target andthe nontarget nucleic acid.

Second, probes should be positioned so as to minimize the stability ofthe probe:nontarget nucleic acid hybrid. This may be accomplished byminimizing the length of perfect complementarity to non-targetorganisms, avoiding G and C rich regions of homology to non-targetsequences, and by positioning the probe to span as many destabilizingmismatches as possible. Whether a probe sequence is useful to detectonly a specific type of organism depends largely on the thermalstability difference between probe:target hybrids and probe:nontargethybrids. In designing probes, the differences in these Tm values shouldbe as large as possible (e.g., at least 2° C. and preferably 5° C.).

The length of the target nucleic acid sequence and, accordingly, thelength of the probe sequence can also be important. In some cases, theremay be several sequences from a particular region, varying in locationand length, which will yield probes with the desired hybridizationcharacteristics. In other cases, one sequence may be significantlybetter than another which differs merely by a single base. While it ispossible for nucleic acids that are not perfectly complementary tohybridize, the longest stretch of perfectly homologous base sequencewill normally primarily determine hybrid stability. Whileoligonucleotide probes of different lengths and base composition may beused, oligonucleotide probes preferred in this invention are betweenabout 10 to 50 bases in length and are sufficiently homologous to thetarget nucleic acid.

Third, regions of the rRNA which are known to form strong internalstructures inhibitory to hybridization are less preferred. Likewise,probes with extensive self-domplementarity should be avoided.

As explained above, hybridization is the association of two singlestrands of complementary nucleic acid to form a hydrogen bonded doublestrand. It is implicit that if one of the two strands is wholly orpartially involved in a hybrid that it will be less able to participatein formation of a new hybrid. In the case of rRNA, the molecule is knownto form very stable intra-molecular hybrids. By designing a probe sothat a substantial portion of the sequence of interest is singlestranded, the rate and extent of hybridization may be greatly increased.If the target is the genomic sequence corresponding to the rRNA then itwill naturally occur in a double stranded form, this is also the casewith the product of the polymerase chain reaction (PCR). These doublestranded targets are naturally inhibitory to hybridization with a probe.Finally, there can be intramolecular and intermolecular hybrids formedwithin a probe if there is sufficient self complementarity. Suchstructures can be avoided through careful probe design. Computerprograms are available to search for this type of interaction.

Once a presumptive unique sequence has been identified, a complementaryDNA oligonucleotide is produced. This single stranded oligonucleotidewill serve as the probe in the hybridization reaction. Definedoligonucleotides may be produced by any of several well known methods,including automated solid-phase chemical synthesis usingcyanoethylphosphoramidite precursors. Barone et al., 12 Nucleic AcidsResearch 4051, 1984. Other well-known methods for construction ofsynthetic oligonucleotides may, of course, be employed. Sambrook et al.,2 Molecular Cloning 11 (2d ed. 1989).

Once synthesized, selected oligonucleotide probes may also be labelledby any of several well known methods. Sambrook et al., supra. Usefullabels include radio-isotopes as well as non-radioactive reportinggroups. Isotopic labels include ³ H, ³⁵ S, ³² P , ¹²⁵ I, Cobalt and ¹⁴C. Most methods of isotopic labelling involve the use of enzymes andinclude the known methods of nick translation, end labelling, secondstrand synthesis, and reverse transcription. When using radio-labelledprobes, hybridization can be detected by autoradiography, scintillationcounting, or gamma counting. The detection method selected will dependupon the hybridization conditions and the particular radio-isotope usedfor labelling.

Non-isotopic materials can also be used for labelling, and may beintroduced internally into the sequence or at the end of the sequence.Modified nucleotides may be incorporated enzymatically or chemically andchemical modifications of the probe may be performed during or aftersynthesis of the probe, for example, by the use of non-nucleotide linkergroups. Non-isotopic labels include fluorescent molecules,chemiluminescent molecules, enzymes, cofactors, enzyme substrates,haptens or other ligands. We currently prefer to use acridinium esters.

Following synthesis and purification of a particular oligonucleotidesequence, several procedures may be utilized to determine theacceptability of the final product. The first is polyacrylamide gelelectrophoresis, which is used to determine size. Sambrook et al.,supra. Such procedures are known in the art. In addition topolyacrylamide gel electrophoresis, High Pressure Liquid Chromatography("HPLC") procedures also may be used to determine the size and purity ofthe oligonucleotide product. These procedures are also known to thoseskilled in the art.

It will be appreciated by those skilled in the art that factors whichaffect the thermal stability can affect probe specificity and therefore,must be controlled. Thus, the melting profile, including the meltingtemperature (Tm) of the oligonucleotide/target hybrids should bedetermined. The preferred method is described in Arnold et al.,PCT/US88/03195, filed Sep. 21, 1988, entitled "Homogeneous ProtectionAssay," hereby incorporated by reference herein.

For Tm measurement using a Hybridization Protection Assay (HPA) thefollowing technique is used. A probe:target hybrid is formed in targetexcess in a lithium succinate buffered solution containing lithiumlauryl sulfate. Aliquots of this hybrid are diluted in the hybridizationbuffer and incubated for five minutes at various temperatures startingbelow that of the anticipated Tm (typically 55° C.) and increasing in2-5 degree increments. This solution is then diluted with a mildlyalkaline borate buffer and incubated at a lower temperature (for example50° C.) for ten minutes. Under these conditions the acridinium esterattached to a single stranded probe is hydrolyzed while that attached tohybridized probe is relatively protected from hydrolysis. The amount ofchemiluminescence remaining is proportional to the amount of hybrid, andis measured in a luminometer by addition of hydrogen peroxide followedby alkali. The data is plotted as percent of maximum signal (usuallyfrom the lowest temperature) versus temperature. The Tm is defined asthe point at which 50% of the maximum signal remains.

In addition to the above method, oligonucleotide/target hybrid meltingtemperature may also be determined by isotopic methods well known tothose skilled in the art. It should be noted that the Tm for a givenhybrid will vary depending on the hybridization solution being usedbecause the thermal stability depends upon the concentration ofdifferent salts, detergents, and other solutes which effect relativehybrid stability during thermal denaturation. Sambrook et al., supra.

Rate of hybridization may be measured by determining the C₀ t_(1/2). Therate at which a probe hybridizes to its target is a measure of thethermal stability of the target secondary structure in the probe region.The standard measurement of hybridization rate is the C₀ t_(1/2) whichis measured as moles of nucleotide per liter times seconds. Thus, it isthe concentration of probe times the half-life of hybridization at thatconcentration. This value is determined by hybridizing various amountsof probe to a constant amount of hybrid for a fixed time. For example,0.05 pmol of target is incubated with 0.0012, 0.025, 0.05, 0.1 and 0.2pmol of probe for 30 minutes. The amount of hybrid after 30 minutes ismeasured by HPA as described above. The signal is then plotted as a logof the percent of maximum Relative Light Units (RLU) (from the highestprobe concentration) versus probe concentration (moles of nucleotide perliter). RLU are a measurement of the quantity of photons emitted by thelabelled-probe measured by the luminometer. The C₀ t_(1/2) is foundgraphically from the concentration corresponding to 50% of maximumhybridization multiplied by the hybridization time in seconds. Thesevalues range from 9.0×10⁻⁶ to 9×10⁻⁵ with the preferred values beingless than 3.5×10⁻⁵.

As described by Kohne and Kacian (EP 86304429.3, filed Jun. 10, 1986),hereby incorporated by reference herein) other methods of nucleic acidreassociation can be used.

The following example sets forth synthetic probes complementary to aunique rRNA sequence, or the corresponding gene, from a target organism,Mycobacterium tuberculosis, and their use in a hybridization assay.

EXAMPLE

A probe specific for M. tuberculosis was identified by sequencing with aprimer complementary to the 16S rRNA. The following sequences werecharacterized and shown to be specific for Mycobacterium tuberculosis;(SEQ ID NO: 1) GGTAGCGCTGAGACATATCCTCC, and (SEQ ID NO: 2)CAGAACTCCACACCCCCGAAG. Several phylogenetically near neighbors includingM. kansasii, M. asiaticum and M. avium were used as comparisons with thesequence of M. tuberculosis. SEQ ID NO: 1 is 23 bases in length andhybridizes to the 23S rRNA of M. tuberculosis corresponding to bases270-293 of E. coli. SEQ ID NO: 2 is 21 bases in length and hybridizes tothe 23S rRNA of M. tuberculosis corresponding to bases 1415-1436 of E.coli.

To demonstrate the reactivity and specificity of the probe for M.tuberculosis, it was used in a hybridization assay. The probe was firstsynthesized with a non-nucleotide linker, then labelled with achemiluminescent acridinium ester as described in EPO Patent ApplicationNo. PCT/US88/03361, entitled "Acridinium Ester Labeling and Purificationof Nucleotide Probes filed Oct. 5, 1988. The acridinium ester attachedto unhybridized probe is rendered non-chemiluminescent under mildalkaline conditions, while the acridinium ester attached to hybridizedprobe is relatively resistant. Thus, it is possible to assay forhybridization of acridinium ester-labelled probe by incubation with analkaline buffer, followed by detection of chemiluminescence in aluminometer. Results are given in RLU, the quantity of photons emittedby the labelled-probe measured by the luminometer. The conditions ofhybridization, hydrolysis and detection are described in Arnold, et al.,35 Clin. Chem. 1588, 1989.

Nucleic acid hybridization was enhanced by the use of "Helper Probes" asdisclosed in Hogan et al., U.S. Pat. No. 5,030,557 hereby incorporatedby reference herein. RNA was hybridized to the acridinium ester-labeledprobe in the presence of an unlabeled Helper Probe. The probecorresponding to oligonucleotide SEQ ID NO: 1 with helpers:

(SEQ ID NO: 3) CCGCTAACCACGACACTTTCTGTACTGCCTCTCAGCCG and

(SEQ ID NO: 4) CACAACCCCGCACACACAACCCCTACCCGGTTACCC.

The probe corresponding to oligonucleotide SEQ ID NO: 2 with helpers:(SEQ ID NO: 5)

TGATTCGTCACGGGCGCCCACACACGGGTACGGGAATATCAACCC and

(SEQ ID NO: 6) CTACTACCAGCCGAAGTTCCCACGCAGCCC and

(SEQ ID NO: 7) GGAGTTGATCGATCCGGTTTTGGGTGGTTAGTACCGC and

(SEQ ID NO: 8) GGGGTACGGGCCGTGTGTGTGCTCGCTAGAGGCTTTTCTTGGC.

In the following experiment, RNA released from one colony or >10⁸organisms was assayed. An example of such a method is provided by Murphyet al. (EP 873036412, filed Apr. 24, 1987), hereby incorporated byreference herein. An RLU value greater than 30,000 RLU is a positivereaction; less than 30,000 is a negative reaction.

The following data show that the probes did riot cross react withorganisms from a wide phylogenetic cross section. The samples were alsotested with a Probe (ALL BACT.) which has a very broad specificity toprovide a positive control. A positive signal from this probe providesconfirmation of sample adequacy.

    ______________________________________              RLU                         ALL    NAME        ATCC #   BACT.    PROBE 1                                         PROBE 2    ______________________________________    Mycobacterium                25420    880551   489764 589419    africanum    M. asiaticum                25276    1291076  708    1849    M. avium    25291    966107   615    1749    M. bovis    19210    1564761  1020088                                         717186    M. bovis BCG                35734    1532845  943131 706773    M. chelonae 14472    1581603  641    1320    M. flavescens                14474    237900   842    2001    M. fortuitum                 6841    910478   641    1710    M. gastri   15754    429144   781    2416    M. gordonae 14470    1207443  749    2089    M. haemophilum                29548    709966   1090   3149    M. intracellulare                13950    277790   823    2512    M. kansasii 12478    416752   839    5688    M. malmoense                29571    149699   1176   4060    M. marinum   927     524740   699    3200    M. nonchromogenicum                19530    1541506  832    3303    M. phlei    11758    1273753  717    2286    M. scrofulaceum                19981    801447   1424   5236    M. shimoidei                27962    1609154  719    2650    M. simiae   25275    1571628  841    3152    M. smegmatis                14468    513995   789    2920    M. szulgai  35799    947710   714    2356    M. terrae   15755    480465   1492   7153    M. thermoresistibile                19527    1054152  1436   4113    M. triviale 23292    1016207  1148   4693    M. tuberculosis (avir.)                25177    1067974  767698 620393    M. tuberculosis (vir.)                27294    1543369  1012711                                         652815    M. ulcerans 19423    1401905  2563   5865    M. vaccae   15483    586428   729    3784    M. xenopi   19250    310648   855    3198    Acinetobacter                33604    1393489  1735   9659    calcoaceticus    Actinomadura                19425    572956   4388   5614    madurae    Actinomyces 19411    1768540  1376   2527    pyogenes    Arthrobacter                14358    1542696  721    2126    oxydans    Bacilius subtilis                6051     1441824  2424   2817    Bacteriodes fragilis                23745    1557888  843    8907    Bordetella  10580    1694010  686    4113    bronchiseptica    Branhamella 25238    1615709  1035   7219    catarrhalis    Brevibacterium linens                9172     904166   814    1642    Campylobacter jejuni                33560    1824094  607    3201    Candida albicans                18804    3850     763    2018    Chromobacterium                29094    1560283  993    11823    violaceum    Clostridium innocuum                14501    1571465  577    2072    C. perfringens                13124    1701191  641    5757    Corynebacterium                14665    1616486  801    1865    aquaticum    C. diphtheriae                11913    1464829  682    1475    c. genitalium                33030    108105   1177   1797    C. haemolyticum                9345     1512544  703    1114    C. matruchotii                33806    1871454  659    1967    C. minutissimum                23347    1024206  586    1302    C. pseudo-  10700    1605944  578    1155    diphtheriticum    C. pseudogenitalium                33035    497387   717    1324    C. pseudotuberculosis                19410    1730057  643    2892    C. renale   19412    1467841  544    1743    C. striatum 6940     1560152  602    1386    C. xerosis  373      1211115  651    1556    Deinococcus 35073    1387623  644    1400    radiodurans    Dermatophilus                14637    1551500  810    2075    congolensis    Derxia gumosa                15994    1735694  4676   4797    Erysipelothrix                19414    1623646  564    1180    rhusiopathiae    Escherichia coli                10798    1685941  581    4610    Flavobacterium                13253    1571895  1037   4626    meniningosepticum    Haemophilus 19418    1706963  668    2303    influenzae    Klebsiella pneumoniae                23357    1692364  639    6673    Lactobacillus                4356     226596   780    1619    acidophilus    Legionella  33152    1666343  755    4184    pneuinophila    Microbacterium                8180     620978   514    924    lacticum    Mycoplasma hominis                14027    1305131  496    1410    M. pneumoniae                15531    1605424  481    1428    Neisseria meningitidis                13077    1684295  1531   8802    Nocardia asteriodes                19247    1265198  1037   1938    N. brasiliensis                19296    1483481  759    1737    N. otitidis-caviarum                14629    1462489  813    1791    Nocardiopsis                23218    662986   4052   4960    dassonvillei    Oerskovia turbata                33225    1753101  591    1979    O. xanthineolytica                27402    1712806  721    1639    Paracoccus  17741    958719   771    2910    denitrificans    Proteus mirabilis                25933    1761750  669    2545    Pseudomonas 25330    1730788  1281   6048    aeruginosa    Rahnella aquatilis                33071    1728428  485    2884    Rhodococcus 33611    528199   595    1169    aichiensis    R. aurantiacus                25936    1737076  616    2310    R. bronchialis                25592    1695267  635    1633    R. chubuensis                33609    1079495  599    1262    R. equi     6939     1762242  709    2863    R. obuensis 33610    658848   686    1482    R. sputi    29627    814617   719    1419    Staphylococcus aureus                12598    1687401  636    1434    S. epidermidis                12228    1117790  651    1255    S. mitis    9811     1807598  542    1199    S. pneumoniae                6306     1883301  532    1441    S. pyogenes 19615    1862392  728    1656    Streptomyces griseus                23345    1417914  1737   3378    Vibrio      17802    1767149  752    6429    parahaemolyticus    Yersinia    9610     1769411  662    4255    enterocolitica    ______________________________________

The above data confirm that the novel probes herein disclosed andclaimed are capable of distinguishing members of the Mycobacteriumtuberculosis complex from their known nearest phylogenetic neighbors.

Other embodiments are within the following claims.

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(2) INFORMATION FOR SEQ ID NO:    8:    -      (i) SEQUENCE CHARACTERISTICS:    #            43ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #8:   (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    # 43               GTGT GCTCGCTAGA GGCTTTTCTT GGC    - (2) INFORMATION FOR SEQ ID NO:    9:    -      (i) SEQUENCE CHARACTERISTICS:    #            23ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #9:   (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #                23CGCT ACC    - (2) INFORMATION FOR SEQ ID NO:    10:    -      (i) SEQUENCE CHARACTERISTICS:    #            23ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #10:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #                23AUCC UCC    - (2) INFORMATION FOR SEQ ID NO:    11:    -      (i) SEQUENCE CHARACTERISTICS:    #            23ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #11:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #                23CGCU ACC    - (2) INFORMATION FOR SEQ ID NO:    12:    -      (i) SEQUENCE CHARACTERISTICS:    #            21ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #12:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                TTCT G    - (2) INFORMATION FOR SEQ ID NO:    13:    -      (i) SEQUENCE CHARACTERISTICS:    #            21ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #13:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                CGAA G    - (2) INFORMATION FOR SEQ ID NO:    14:    -      (i) SEQUENCE CHARACTERISTICS:    #            21ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #14:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                UUCU G    - (2) INFORMATION FOR SEQ ID NO:    15:    -      (i) SEQUENCE CHARACTERISTICS:    #            38ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #15:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #     38           CAGA AAGTGTCGTG GTTAGCGG    - (2) INFORMATION FOR SEQ ID NO:    16:    -      (i) SEQUENCE CHARACTERISTICS:    #            36ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #16:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #       36         GTTG TGTGTGCGGG GTTGTG    - (2) INFORMATION FOR SEQ ID NO:    17:    -      (i) SEQUENCE CHARACTERISTICS:    #            38ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #17:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #     38           UUUC UGUACUGCCU CUCAGCCG    - (2) INFORMATION FOR SEQ ID NO:    18:    -      (i) SEQUENCE CHARACTERISTICS:    #            36ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #18:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #       36         CAAC CCCUACCCGG UUACCC    - (2) INFORMATION FOR SEQ ID NO:    19:    -      (i) SEQUENCE CHARACTERISTICS:    #            38ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #19:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #     38           CAGA AAGUGUCGUG GUUAGCGG    - (2) INFORMATION FOR SEQ ID NO:    20:    -      (i) SEQUENCE CHARACTERISTICS:    #            36ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #20:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #       36         GUUG UGUGUGCGGG GUUGUG    - (2) INFORMATION FOR SEQ ID NO:    21:    -      (i) SEQUENCE CHARACTERISTICS:    #            45ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #21:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #45                ACCC GTGTGTGGGC GCCCGTGACG AATCA    - (2) INFORMATION FOR SEQ ID NO:    22:    -      (i) SEQUENCE CHARACTERISTICS:    #            30ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #22:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #           30     TCGG CTGGTAGTAG    - (2) INFORMATION FOR SEQ ID NO:    23:    -      (i) SEQUENCE CHARACTERISTICS:    #            37ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #23:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #      37          AAAA CCGGATCGAT CAACTCC    - (2) INFORMATION FOR SEQ ID NO:    24:    -      (i) SEQUENCE CHARACTERISTICS:    #            43ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #24:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    # 43               AGCG AGCACACACA CGGCCCGTAC CCC    - (2) INFORMATION FOR SEQ ID NO:    25:    -      (i) SEQUENCE CHARACTERISTICS:    #            45ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #25:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #45                CCCA CACACGGGUA CGGGAAUAUC AACCC    - (2) INFORMATION FOR SEQ ID NO:    26:    -      (i) SEQUENCE CHARACTERISTICS:    #            30ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #26:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #           30     UUCC CACGCAGCCC    - (2) INFORMATION FOR SEQ ID NO:    27:    -      (i) SEQUENCE CHARACTERISTICS:    #            37ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #27:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #      37          GUUU UGGGUGGUUA GUACCGC    - (2) INFORMATION FOR SEQ ID NO:    28:    -      (i) SEQUENCE CHARACTERISTICS:    #            43ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #28:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    # 43               GUGU GCUCGCUAGA GGCUUUUCUU GGC    - (2) INFORMATION FOR SEQ ID NO:    29:    -      (i) SEQUENCE CHARACTERISTICS:    #            45ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #29:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #45                ACCC GUGUGUGGGC GCCCGUGACG AAUCA    - (2) INFORMATION FOR SEQ ID NO:    30:    -      (i) SEQUENCE CHARACTERISTICS:    #            30ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #30:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #           30     UCGG CUGGUAGUAG    - (2) INFORMATION FOR SEQ ID NO:    31:    -      (i) SEQUENCE CHARACTERISTICS:    #            37ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #31:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    #      37          AAAA CCGGAUCGAU CAACUCC    - (2) INFORMATION FOR SEQ ID NO:    32:    -      (i) SEQUENCE CHARACTERISTICS:    #            43ENGTH:    #              nucleic acid    #        singleTRANDEDNESS:    #           linearLOGY:    #32:  (ii) SEQUENCE DESCRIPTION: SEQ ID NO:    # 43               AGCG AGCACACACA CGGCCCGUAC CCC    __________________________________________________________________________

What is claim is:
 1. A hybridization assay probe for detecting thepresence of Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium bovis BCG and Mycobacterium africanum nucleic acid,comprising an oligonucleotide which is 15 to 100 nucleotides in lengthcontaining at least 14 out of 17 contiguous bases perfectlycomplementary to a nucleic acid sequence selected from the groupconsisting of:SEQ ID NO 1: GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 2:CAGAACTCCACACCCCCGAAG, SEQ ID NO 10: GGUAGCGCUGAGACAUAUCCUCC, SEQ ID NO11: GGAGGAUAUGUCUCAGCGCUACC, SEQ ID NO 13: CAGAACUCCACACCCCCGAAG, andSEQ ID NO 14: CUUCGGGGGUGUGGAGUUCUG;wherein said oligonucleotidehybridizes to Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium bovis BCG and Mycobacterium africanum nucleic acid underhigh stringency hybridization conditions, wherein under said conditionssaid probe can distinguish Mycobacterium tuberculosis, Mycobacteriumbovis, Mycobacterium bovis BCG and Mycobacterium africanum nucleic acid,from Mycobacterium avium, Mycobacterium asiaticum, and Mycobacteriumkansasii nucleic acid.
 2. The probe of claim 1, wherein said nucleicacid sequence is selected from the group consisting of: SEQ ID NO 2:CAGAACTCCACACCCCCGAAG, SEQ ID NO 13: CAGAACUCCACACCCCCGAAG, and SEQ IDNO 14: CUUCGGGGGUGUGGAGUUCUG.
 3. The probe of claim 2, wherein saidoligonucleotide comprises a nucleotide sequence selected from the groupconsisting of:SEQ ID NO 2: CAGAACTCCACACCCCCGAAG, SEQ ID NO 12:CTTCGGGGGTGTGGAGTTCTG, SEQ ID NO 13: CAGAACUCCACACCCCCGAAG, and SEQ IDNO 14: CUUCGGGGGUGUGGAGUUCUG.
 4. The probe of claim 3, wherein saidoligonucleotide is 21 to 50 bases in length.
 5. The probe of claim 3,wherein said probe consists of said nucleotide sequence and one or moreoptionally present detectable labels, wherein said optionally presentdetectable labels may or may not be present.
 6. The probe of claim 5,wherein said probe contains a detectable label selected from the groupconsisting of: radioisotope, fluorescent molecule, chemiluminescentmolecule, enzyme, cofactor, enzyme substrate, and hapten.
 7. The probeof claim 6, wherein said detectable label is an acridinium ester.
 8. Theprobe of claim 1, wherein said nucleic acid sequence is selected fromthe group consisting of:SEQ ID NO 1: GGTAGCGCTGAGACATATCCTCC, SEQ ID NO10: GGUAGCGCUGAGACAUAUCCUCC, and SEQ ID NO 11: GGAGGAUAUGUCUCAGCGCUACC.9. The probe of claim 8, wherein said oligonucleotide comprises anucleotide sequence selected from the group consisting of:SEQ ID NO 1:GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 9: GGAGGATATGTCTCAGCGCTACC, SEQ ID NO10: GGUAGCGCUGAGACAUAUCCUCC, and SEQ ID NO 11: GGAGGAUAUGUCUCAGCGCUACC.10. The probe of claim 9, wherein said oligonucleotide is 23 to 50 basesin length.
 11. The probe of claim 9, wherein said probe consists of saidnucleotide sequence and one or more optionally present detectablelabels, wherein said optionally present detectable labels may or may notbe present.
 12. The probe of claim 11, wherein said probe contains adetectable label selected from the group consisting of: radioisotope,fluorescent molecule, chemiluminescent molecule, enzyme, cofactor,enzyme substrate, and hapten.
 13. The probe of claim 12, wherein saiddetectable label is an acridinium ester.
 14. A nucleic acid hybridcomprising:a) a hybridization assay probe for detecting the presence ofMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCGand Mycobacterium africanum nucleic acid, comprising an oligonucleotidewhich is 15 to 100 nucleotides in length containing at least 14 out of17 contiguous bases perfectly complementary to a target nucleic acidsequence selected from the group consisting of: SEQ ID NO 1:GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 2: CAGAACTCCACACCCCCGAAG, SEQ ID NO10: GGUAGCGCUGAGACAUAUCCUCC, SEQ ID NO 11: GGAGGAUAUGUCUCAGCGCUACC, SEQID NO 13: CAGAACUCCACACCCCCGAAG, and SEQ ID NO 14:CUUCGGGGGUGUGGAGUUCUG;wherein said oligonucleotide hybridizes toMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCGand Mycobacterium africanum nucleic acid under high stringencyhybridization conditions, wherein under said conditions said probe candistinguish Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium bovis BCG and Mycobacterium africanum nucleic acid, fromMycobacterium avium, Mycobacterium asiaticum, and Mycobacterium kansasiinucleic acid; and b) Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium bovis BCG or Mycobacterium africanum nucleic acidcontaining a complementary region, said complementary region selectedfrom the group consisting of:23S rRNA corresponding to bases 270-293 ofE. coli, rDNA gene for 23S rRNA corresponding to bases 270-293 of E.coli, 23S rRNA corresponding to bases 1415-1436 of E. coli, and rDNAgene for 23S rRNA corresponding to bases 1415-1436 of E. coli.
 15. Thehybrid of claim 14, wherein said target nucleic acid sequence isselected from the group consisting of:SEQ ID NO 2:CAGAACTCCACACCCCCGAAG, SEQ ID NO 13: CAGAACUCCACACCCCCGAAG, and SEQ IDNO 14: CUUCGGGGGUGUGGAGUUCUG.
 16. The hybrid of claim 15, wherein saidoligonucleotide is 21 to 50 bases in length and comprises a nucleotidesequence selected from the group consisting of:SEQ ID NO 2:CAGAACTCCACACCCCCGAAG, SEQ ID NO 12: CTTCGGGGGTGTGGAGTTCTG, SEQ ID NO13: CAGAACUCCACACCCCCGAAG, and SEQ ID NO 14: CUUCGGGGGUGUGGAGUUCUG. 17.The probe of claim 16, wherein said probe consists of said nucleotidesequence and one or more optionally present detectable labels, whereinsaid optionally present detectable labels may or may not be present. 18.The hybrid of claim 14, wherein said target nucleic acid sequence isselected from the group consisting of:SEQ ID NO 1:GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 10: GGUAGCGCUGAGACAUAUCCUCC, and SEQID NO 11: GGAGGAUAUGUCUCAGCGCUACC.
 19. The hybrid of claim 18, whereinsaid oligonucleotide is 23 to 50 bases in length and comprises anucleotide sequence selected from the group consisting of:SEQ ID NO 1:GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 9: GGAGGATATGTCTCAGCGCTACC, SEQ ID NO10: GGUAGCGCUGAGACAUAUCCUCC, and SEQ ID NO 11: GGAGGAUAUGUCUCAGCGCUACC.20. The hybrid of claim 19, wherein said probe consists of saidnucleotide sequence and one or more optionally present detectablelabels, wherein said optionally present detectable labels may or may notbe present.
 21. A method for determining if at least one ofMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCGand Mycobacterium africanum may be present in a sample comprising thesteps of:a) contacting said sample under high stringency hybridizationconditions with a hybridization assay probe for detecting the presenceof Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovisBCG and Mycobacterium africanum nucleic acid, said probe comprising anoligonucleotide containing at least 14 out of 17 contiguous basesperfectly complementary to a nucleic acid sequence selected from thegroup consisting of:SEQ ID NO 1: GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 2:CAGAACTCCACACCCCCGAAG, SEQ ID NO 10: GGUAGCGCUGAGACAUAUCCUCC, SEQ ID NO11: GGAGGAUAUGUCUCAGCGCUACC, SEQ ID NO 13: CAGAACUCCACACCCCCGAAG, andSEQ ID NO 14: CUUCGGGGGUGUGGAGUUCUG;wherein said oligonucleotidehybridizes to Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium bovis BCG and Mycobacterium africanum nucleic acid underhigh stringency hybridization conditions, wherein under said conditionssaid probe can distinguish Mycobacterium tuberculosis, Mycobacteriumbovis, Mycobacterium bovis BCG and Mycobacterium africanum nucleic acid,from Mycobacterium avium, Mycobacterium asiaticum, and Mycobacteriumkansasii nucleic acid; and b) detecting hybridization of said probe tonucleic acid present in said sample under said high stringencyhybridization conditions as an indication that at least one ofMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCGand Mycobacterium africanum may be present.
 22. The method of claim 21,wherein said nucleic acid sequence is selected from the group consistingof:SEQ ID NO 2: CAGAACTCCACACCCCCGAAG, SEQ ID NO 13:CAGAACUCCACACCCCCGAAG, and SEQ ID NO 14: CUUCGGGGGUGUGGAGUUCUG.
 23. Themethod of claim 22, wherein said oligonucleotide comprises a nucleotidesequence selected from the group consisting of:SEQ ID NO 2:CAGAACTCCACACCCCCGAAG, SEQ ID NO 12: CTTCGGGGGTGTGGAGTTCTG, SEQ ID NO13: CAGAACUCCACACCCCCGAAG, and SEQ ID NO 14: CUUCGGGGGUGUGGAGUUCUG. 24.The method of claim 23, wherein said oligonucleotide is 21 to 50 basesin length.
 25. The method of claim 23, wherein said probe consists ofsaid nucleotide sequence and one or more optionally present detectablelabels, wherein said optionally present detectable labels may or may notbe present.
 26. The method of claim 21, wherein said nucleic acidsequence is selected from the group consisting of:SEQ ID NO 1:GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 10: GGUAGCGCUGAGACAUAUCCUCC, and SEQID NO 11: GGAGGAUAUGUCUCAGCGCUACC.
 27. The method of claim 26, whereinsaid oligonucleotide comprises a nucleotide sequence selected from thegroup consisting of:SEQ ID NO 1: GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 9:GGAGGATATGTCTCAGCGCTACC, SEQ ID NO 10: GGUAGCGCUGAGACAUAUCCUCC, and SEQID NO 11: GGAGGAUAUGUCUCAGCGCUACC.
 28. The method of claim 27, whereinsaid oligonucleotide is 23 to 50 bases in length.
 29. The method ofclaim 27, wherein said probe consists of said nucleotide sequence andone or more optionally present detectable labels, wherein saidoptionally present detectable labels mav or may not be present.
 30. Acomposition comprising:a) a hybridization assay probe for detecting thepresence of Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium bovis BCG and Mycobacterium africanum nucleic acid, saidprobe comprising an oligonucleotide which is 15 to 100 nucleotides inlength containing at least 14 out of 17 contiguous bases perfectlycomplementary to a hybridization assay probe target nucleic acidsequence selected from the group consisting of:SEQ ID NO 1:GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 2: CAGAACTCCACACCCCCGAAG, SEQ ID NO10: GGUAGCGCUGAGACAUAUCCUCC, SEQ ID NO 11: GGAGGAUAUGUCUCAGCGCUACC, SEQID NO 13: CAGAACUCCACACCCCCGAAG, and SEQ ID NO 14:CUUCGGGGGUGUGGAGUUCUG;wherein said oligonucleotide hybridizes toMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCGand Mycobacterium africanum nucleic acid under said high stringencyhybridization conditions, wherein under said conditions said probe candistinguish Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium bovis BCG and Mycobacterium africanum nucleic acid, fromMycobacterium avium, Mycobacterium asiaticum, and Mycobacterium kansasiinucleic acid; and at least one helper probe comprising a helper probenucleotide sequence selected from the group consisting of:SEQ ID NO: 3CCGCTAACCA CGACACTTTC TGTACTGCCT CTCAGCCG, SEQ ID NO: 4 CACAACCCCGCACACACAAC CCCTACCCGG TTACCC, SEQ ID NO: 5 TGATTCGTCA CGGGCGCCCACACACGGGTA CGGGAATATC AACCC, SEQ ID NO: 6 CTACTACCAG CCGAAGTTCCCACGCAGCCC, SEQ ID NO: 7 GGAGTTGATC GATCCGGTTT TGGGTGGTTA GTACCGC, SEQID NO: 8 GGGGTACGGG CCGTGTGTGT GCTCGCTAGA GGCTTTTCTT GGC, SEQ ID NO: 15CGGCTGAGAG GCAGTACAGA AAGTGTCGTG GTTAGCGG, SEQ ID NO: 16 GGGTAACCGGGTAGGGGTTG TGTGTGCGGG GTTGTG, SEQ ID NO: 17 CCGCUAACCA CGACACUUUCUGUACUGCCU CUCAGCCG, SEQ ID NO: 18 CACAACCCCG CACACACAAC CCCUACCCGGUUACCC, SEQ ID NO: 19 CGGCUGAGAG GCAGUACAGA AAGUGUCGUG GUUAGCGG, SEQ IDNO: 20 GGGUAACCGG GUAGGGGUUG UGUGUGCGGG GUUGUG, SEQ ID NO: 21 GGGTTGATATTCCCGTACCC GTGTGTGGGC GCCCGTGACG AATCA, SEQ ID NO: 22 GGGCTGCGTGGGAACTTCGG CTGGTAGTAG, SEQ ID NO: 23 GCGGTACTAA CCACCCAAAA CCGGATCGATCAACTCC, SEQ ID NO: 24 GCCAAGAAAA GCCTCTAGCG AGCACACACA CGGCCCGTAC CCC,SEQ ID NO: 25 UGAUUCGUCA CGGGCGCCCA CACACGGGUA CGGGAAUAUC AACCC, SEQ IDNO: 26 CUACUACCAG CCGAAGUUCC CACGCAGCCC, SEQ ID NO: 27 GGAGUUGAUCGAUCCGGUUU UGGGUGGUUA GUACCGC, SEQ ID NO: 28 GGGGUACGGG CCGUGUGUGUGCUCGCUAGA GGCUUUUCUU GGC, SEQ ID NO: 29 GGGUUGAUAU UCCCGUACCCGUGUGUGGGC GCCCGUGACG AAUCA, SEQ ID NO: 30 GGGCUGCGUG GGAACUUCGGCUGGUAGUAG, SEQ ID NO: 31 GCGGUACUAA CCACCCAAAA CCGGAUCGAU CAACUCC, andSEQ ID NO: 32 GCCAAGAAAA GCCUCUAGCG AGCACACACA CGGCCCGUAC CCC.
 31. Thecomposition of claim 30, wherein said hybridization assay probe targetnucleic acid sequence is selected from the group consisting of:SEQ ID NO1: GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 10: GGUAGCGCUGAGACAUAUCCUCC, andSEQ ID NO 11: GGAGGAUAUGUCUCAGCGCUACC; and said at least one helperprobe comprises a helper probe nucleotide sequence selected from thegroup consisting of:SEQ ID NO: 3 CCGCTAACCA CGACACTTTC TGTACTGCCTCTCAGCCG, SEQ ID NO: 4 CACAACCCCG CACACACAAC CCCTACCCGG TTACCC, SEQ IDNO: 15 CGGCTGAGAG GCAGTACAGA AAGTGTCGTG GTTAGCGG, SEQ ID NO: 16GGGTAACCGG GTAGGGGTTG TGTGTGCGGG GTTGTG, SEQ ID NO: 17 CCGCUAACCACGACACUUUC UGUACUGCCU CUCAGCCG, and SEQ ID NO: 18 CACAACCCCG CACACACAACCCCUACCCGG UUACCC.
 32. The composition of claim 31, wherein saidhybridization assay probe is 23 to 50 bases in length and comprises ahybridization assay probe nucleotide sequence selected from the groupconsisting of:SEQ ID NO 1: GGTAGCGCTGAGACATATCCTCC, SEQ ID NO 9:GGAGGATATGTCTCAGCGCTACC, SEQ ID NO 10: GGUAGCGCUGAGACAUAUCCUCC, and SEQID NO 11: GGAGGAUAUGUCUCAGCGCUACC.
 33. The composition of claim 32,wherein said hybridization assay probe consists of said hybridizationassay probe nucleotide sequence which is labeled, and said at least onehelper probe consists of said helper probe nucleotide sequence.
 34. Thecomposition of claim 33, wherein said hybridization assay probenucleotide sequence is either SEQ ID NO 1: GGTAGCGCTGAGACATATCCTCC orSEQ ID NO 10: GGUAGCGCUGAGACAUAUCCUCC; and said at least one helperprobe consists of a first helper probe of SEQ ID NO: 3 CCGCTAACCACGACACTTTC TGTACTGCCT CTCAGCCG, and a second helper probe of SEQ ID NO:4 CACAACCCCG CACACACAAC CCCTACCCGG TTACCC.
 35. The composition of claim33, wherein said hybridization assay probe nucleotide sequence is eitherSEQ ID NO 9: GGAGGATATGTCTCAGCGCTACC, or SEQ ID NO 11:GGAGGAUAUGUCUCAGCGCUACC; and said at least one helper probe consists ofa first helper probe of SEQ ID NO: 15 CGGCTGAGAG GCAGTACAGA AAGTGTCGTGGTTAGCGG, and a second helper probe of SEQ ID NO: 16 GGGTAACCGGGTAGGGGTTG TGTGTGCGGG GTTGTG.
 36. The composition of claim 30, whereinsaid hybridization assay probe target nucleic acid sequence is selectedfrom the group consisting of:SEQ ID NO 2: CAGAACTCCACACCCCCGAAG, SEQ IDNO 13: CAGAACUCCACACCCCCGAAG, and SEQ ID NO 14: CUUCGGGGGUGUGGAGUUCUG;and said at least one helper probe comprises a helper probe nucleotidesequence selected from the group consisting of:SEQ ID NO: 5 TGATTCGTCACGGGCGCCCA CACACGGGTA CGGGAATATC AACCC, SEQ ID NO: 6 CTACTACCAGCCGAAGTTCC CACGCAGCCC, SEQ ID NO: 7 GGAGTTGATC GATCCGGTTT TGGGTGGTTAGTACCGC, SEQ ID NO: 8 GGGGTACGGG CCGTGTGTGT GCTCGCTAGA GGCTTTTCTT GGC,SEQ ID NO: 25 UGAUUCGUCA CGGGCGCCCA CACACGGGUA CGGGAAUAUC AACCC, SEQ IDNO: 26 CUACUACCAG CCGAAGUUCC CACGCAGCCC, SEQ ID NO: 27 GGAGUUGAUCGAUCCGGUUU UGGGUGGUUA GUACCGC, and SEQ ID NO: 28 GGGGUACGGG CCGUGUGUGUGCUCGCUAGA GGCUUUUCUU GGC.
 37. The composition of claim 36, wherein saidhybridization assay probe is 23 to 50 bases in length and comprises ahybridization assay probe nucleotide sequence selected from the groupconsisting of:SEQ ID NO 2: CAGAACTCCACACCCCCGAAG, SEQ ID NO 12:CTTCGGGGGTGTGGAGTTCTG, SEQ ID NO 13: CAGAACUCCACACCCCCGAAG, and SEQ IDNO 14: CUUCGGGGGUGUGGAGUUCUG.
 38. The composition of claim 37, whereinsaid hybridization assay probe consists of said hybridization assayprobe nucleotide sequence which is labeled and said at least one helperprobe consists of said helper probe nucleotide sequence.
 39. Thecomposition of claim 38, wherein said hybridization assay probenucleotide sequence is either SEQ ID NO 2: CAGAACTCCACACCCCCGAAG, or SEQID NO 13: CAGAACUCCACACCCCCGAAG; and said at least one helper probeconsists of a first helper probe of SEQ ID NO: 5 TGATTCGTCA CGGGCGCCCACACACGGGTA CGGGAATATC AACCC, a second helper probe of SEQ ID NO: 6CTACTACCAG CCGAAGTTCC CACGCAGCCC, a third helper probe of SEQ ID NO: 7GGAGTTGATC GATCCGGTTT TGGGTGGTTA GTACCGC, and a fourth helper probe ofSEQ ID NO: 8 GGGGTACGGG CCGTGTGTGT GCTCGCTAGA GGCTTTTCTT GGC.
 40. Amethod for determining if at least one of Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium bovis BCG and Mycobacterium africanummay be present in a sample comprising the steps of:a) providing to saidsample a nucleic acid hybridization assay probe which underhybridization buffer conditions employed during hybridization assayconditions hybridizes to Mycobacterium tuberculosis, Mycobacteriumbovis, Mycobacterium bovis BCG and Mycobacterium africanum nucleic acidin a region corresponding to nucleotide position 270-292 of E. coli 16SrRNA or 1415-1435 of E. coli 23S rRNA, or the complements thereof, toform a probe:target hybrid with a Tm which is at least 2° C. greaterthan a probe:non-target hybrid formed with said region present innucleic acid of Mycobacterium avium, Mycobacterium asiaticum, andMycobacterium kansasii, and b) detecting whether probe:target hybridshave formed under said hybridization assay conditions as an indicationthat at least one of Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium bovis BCG and Mycobacterium africanum may be present,wherein under said hybridization assay conditions detectableprobe:non-target hybrids do not form.
 41. The method of claim 40,wherein said region corresponds to nucleotide position 270-292 of E.coli 16S rRNA, or the complement thereof.
 42. The method of claim 41,wherein under said hybridization buffer conditions employed duringhybridization assay conditions said probe forms a probe:target hybridwith a Tm which is at least 5° C. greater than a probe:non-target hybridformed with said region present in nucleic acid of Mycobacterium avium,Mycobacterium asiaticum, and Mycobacterium kansasii.
 43. The method ofclaim 40, wherein said region corresponds to nucleotide position1415-1435 of E. coli 23S rRNA, or the complement thereof.
 44. The methodof claim 43, wherein under said hybridization buffer conditions employedduring hybridization assay conditions said probe forms a probe:targethybrid with a Tm which is at least 5° C. greater than a probe:non-targethybrid formed with said region present in nucleic acid of Mycobacteriumavium, Mycobacterium asiaticum, and Mycobacterium kansasii.
 45. A methodfor determining if at least one of Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium bovis BCG and Mycobacterium africanummay be present in a sample comprising the steps of: a) exposing saidsample to a hybridization assay means which detects whether aMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCGand Mycobacterium africanum nucleic acid target region located in aposition corresponding to nucleotide position 270-292 of E. coli 16SrRNA or 1415-1435 of E. coli 23S rRNA, or the complements thereof, ispresent in said sample, andb) determining whether said means detects thepresence of said target region in said sample as an indication that atleast one of Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium bovis BCG and Mycobacterium africanum may be present insaid sample, provided that said means distinguishes Mycobacteriumtuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG andMycobacterium africanum nucleic acid, from Mycobacterium avium,Mycobacterium asiaticum, and Mycobacterium kansasii nucleic acid. 46.The method of claim 45, wherein said target region corresponds tonucleotide position 270-292 of E. coli 16S rRNA, or the complementthereof.
 47. The method of claim 45, wherein said target regioncorresponds to nucleotide position 1415-1435 of E. coli 23S rRNA, or thecomplement thereof.